TEST METHODS

Sequencing with CNV

Pricing Comments

Our favored testing approach is exome based NextGen sequencing with CNV analysis. This will allow cost effective reflexing to PGxome or other exome based tests. However, if full gene Sanger sequencing is desired for STAT turnaround time, insurance, or other reasons, please see link below for Test Code, pricing, and turnaround time information. If the Sanger option is selected, CNV detection may be ordered through Test #600.

Targeted Testing

Turnaround Time

The great majority of tests are completed within 26 days.

Clinical Sensitivity

Pathogenic mutations in KIF7 were found in all 14 patients with ACLS (Putoux et al. 2011; Putoux et al. 2012). The clinical sensitivity of KIF7 mutations in patients with HLS and JS is unknown due to a small number of individual cases reported (Putoux et al. 2011; Dafinger et al. 2011).

No gross deletions/duplications have been reported in KIF7 (Human Gene Mutation Database).

See More

See Less

Clinical Features

Joubert Syndrome (JS) is marked by hypotonia, abnormal ocular movements, neonatal respiratory difficulties, mental retardation, hypoplasia of the cerebellar vermis, and malformation of the brainstem. The brain malformations lead to the "molar tooth sign" on cranial MRI, which is the hallmark clinical feature of JS. Other variable JS features include cystic kidneys, nephronophthisis, retinal dystrophy, ocular coloboma, occipital encephalocele, polydactyly, ataxia, and hepatic fibrosis. For more information, see Parisi and Glass (2013) and Parisi et al. (2007). Fetal hydrolethalus syndrome (HLS) and acrocallosal syndrome (ACLS) have many clinical features that overlap with Joubert syndrome, such as midline-hindbrain malformations, facial abnormalities, and polydactyly (Putoux et al. 2011). Hydrolethalus syndrome is a lethal disorder characterized by hydrocephaly or anencephaly with a keyhole foramen magnum and, polydactyly of the hands and feet (Putoux et al. 2011). ACLS is characterized by macrocephaly, intellectual disabilities, hypertelorism, and polydactyly of the hands and feet.

Genetics

JS, HLS and ACLS are inherited in an autosomal recessive manner. Homozygous or compound heterozygous mutations in the KIF7 gene are documented to cause JS, HLS and ACLS (Putoux et al. 2011; Putoux et al. 2012; Dafinger et al. 2011). JS is a heterogeneous genetic disorder caused by mutations in at least 20 genes, including KIF7 (Parisi and Glass 2013). KIF7 encodes for a cilia-associated protein that is a negative regulator of sonic hedgehog signaling (Liem et al. 2009). The majority of pathogenic variants reported to date are chain terminating mutations (nonsense, splicing and frameshift), although missense mutations in KIF7 have also been found in patients with ACLS and HLS.

Testing Strategy

For this Next Generation Sequencing (NGS) test, sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for regions not captured or with insufficient number of sequence reads.

For Sanger sequencing, polymerase chain reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

This test provides full coverage of all coding exons of the KIF7 gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.

Since this test is performed using exome capture probes, a reflex to any of our exome based tests is available (PGxome, PGxome Custom Panels).

Indications for Test

Candidates for this test are patients with symptoms consistent with ACLS, HS, or JS, and family members of patients who have known mutations.

TEST METHODS

Exome Sequencing with CNV Detection

Test Procedure

For the PGxome we use Next Generation Sequencing (NGS) technologies to cover the coding regions of targeted genes plus ~10 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from patient specimens. Patient DNA corresponding to these regions is captured using Agilent Clinical Research Exome hybridization probes. Captured DNA is sequenced on the NovaSeq 6000 using 2x150 bp paired-end reads (Illumina, San Diego, CA, USA). The following quality control metrics are generally achieved: >97% of target bases are covered at >20x, and mean coverage of target bases >120x. Data analysis and interpretation is performed by the internally developed software Titanium-Exome. In brief, the output data from the NovaSeq 6000 is converted to fastqs by Illumina Bcl2Fastq, and mapped by BWA. Variant calls are made by the GATK Haplotype caller and annotated using in house software and SnpEff. Variants are filtered and annotated using VarSeq (www.goldenhelix.com).

For Sanger sequencing, polymerase chain reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

Analytical Validity

NextGen Sequencing: As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.

In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.

Copy Number Variant Analysis: The PGxome test detects most larger deletions and duplications including intragenic CNVs and large cytogenetic events; however aberrations in a small percentage of regions may not be accurately detected due to sequence paralogy (e.g., pseudogenes, segmental duplications), sequence properties, deletion/duplication size (e.g., 1-3 exons vs. 4 or more exons), and inadequate coverage. In general, sensitivity for single, double, or triple exon CNVs is ~70% and for CNVs of four exon size or larger is >95%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.

Analytical Limitations

Interpretation of the test results is limited by the information that is currently available. Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

When sequencing does not reveal any heterozygous differences from the reference sequence, we cannot be certain that we were able to detect both patient alleles.

For technical reasons, the PGxome test is not 100% sensitive. Some exons cannot be efficiently captured, and some genes cannot be accurately sequenced because of the presence of multiple copies in the genome. Therefore, a small fraction of sequence variants will not be detected.

In most cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants due to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient's nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during amplification.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes if taken from whole blood). Test reports contain no information about the DNA sequence in other cell-types.

We cannot be certain that the reference sequences are correct.

Balanced translocations or inversions are only rarely detected.

Certain types of sex chromosome aneuploidy may not be detected.

Our ability to detect CNVs due to somatic mosaicism is limited.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics. However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.

A negative finding does not rule out a genetic diagnosis.

Genetic counseling to help to explain test results to the patients and to discuss reproductive options is recommended.

Ship blood tubes at room temperature in an insulated container. Do not freeze blood.

During hot weather, include a frozen ice pack in the shipping container.
Place a paper towel or other thin material between the ice pack and the blood tube.

In cold weather, include an unfrozen ice pack in the shipping container as insulation.

At room temperature, blood specimen is stable for up to 48 hours.

If refrigerated, blood specimen is stable for up to one week.

Label the tube with the patient name, date of birth and/or ID number.

DNA

(Delivery accepted Monday - Saturday)

Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.

For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.

DNA may be shipped at room temperature.

Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.

We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.

CELL CULTURE

(Delivery preferred Monday - Thursday)

PreventionGenetics should be notified in advance of arrival of a cell culture.

Culture and send at least two T25 flasks of confluent cells.

Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.

Send specimens in insulated, shatterproof container overnight.

Cell cultures may be shipped at room temperature or refrigerated.

Label the flasks with the patient name, date of birth, and/or ID number.

We strongly recommend maintaining a local back-up culture. We do not culture cells.

We use cookies and other tracking technologies to improve your browsing experience on our website, to analyze our website traffic, and to understand where our visitors are coming from. By browsing our website, you consent to our use of cookies and other tracking technologies.